Photosensitized light-induced damage of IRBP (interphotoreceptor retinoid-binding protein): effects on binding properties.

PURPOSE: To determine if IRBP (interphotoreceptor retinoid-binding protein) is damaged following irradiation by visible light in the presence of bound all-trans retinal. METHODS: Following irradiation of the IRBP-all-trans retinal complex, the retinal was removed and damage to IRBP measured as loss of titratable thiol groups, loss of tryptophan fluorescence, and changes in retinol-binding-induced fluorescence. RESULTS: IRBP irradiated by itself showed only minimal loss of tryptophan fluorescence; this loss was substantially increased by irradiation in the presence of all-trans retinal. Thiol groups and retinol-binding activity were also shown to be reduced. The damage to IRBP seemed to involve photosensitization by the all-trans retinal, which was in turn protected from bleaching by the IRBP. The binding affinity was shown to be reduced ten-fold following irradiation. CONCLUSION: In the eye, IRBP can stabilise vitamin A and debatably may be responsible for transport of different forms of vitamin A between the photoreceptor cells and pigment epithelium. If this is the case, it would play a key role in rhodopsin regeneration after bleaching. IRBP also appears to be necessary to sustain photoreceptor cells. Light was shown to cause photosensitized damage to IRBP, and thus might impair the regeneration process and photoreceptor viability.

Photoreceptor rim protein: partial sequences of cDNA show a high degree of similarity to ABC transporters.

PURPOSE: To isolate and sequence cDNA for bovine rim protein, a large membrane-bound glycoprotein found in photoreceptor outer segments. METHODS: Bovine rim protein was N-terminally sequenced (22 residues) and fragments were prepared by partial proteolysis. Two internal sequences of 21 and 18 amino acid residues were obtained from 35 kDa and 32 kDa fragments, respectively. Sense and anti-sense oligonucleotide primers were constructed, based on the peptide sequences derived from the 35 kDa and 32 kDa fragments, respectively, and the polymerase chain reaction (PCR) was used to amplify a 150 bp sequence from bovine retinal cDNA. RESULTS: The amplified sequence coded for the remainder of the peptide sequence determined from the 35 kDa fragment, which was not present in the primer, confirming that it was derived from the rim protein. The 150 bp sequence was translated to give a 50 amino acid peptide. Part of this peptide was compared with Dna sequence databases using the TFastA program, which found 94.6% identity with an EST derived from human retina and 86.1% identity to the mouse abc1 transporter. CONCLUSIONS: It is proposed that rim protein is a member of the ATP transporter family of proteins. It may be involved in transport of molecules involved in visual transduction across the photoreceptor disk membrane.

Linkage of a medium sized Scottish autosomal dominant retinitis pigmentosa family to chromosome 7q.

Retinitis pigmentosa is a group of hereditary retinopathies which is both clinically and genetically heterogeneous. Autosomal dominant (ADRP), autosomal recessive (ARRP), and X linked recessive (XLRP), as well as digenic forms of inheritance have been reported. ADRP has been linked to 3q, 6p, 7p, 7q, 8cen, 17p, 17q, and 19q. Three unrelated ADRP families have been reported to show linkage to 7q. We tested a Scottish ADRP family with microsatellite markers mapping within the 7q31-q35 region, and found three markers (D7S487, D7S514, D7S530) showing statistically significant evidence of linkage. A maximum two point lod score of 3.311 at 0% recombination was obtained for D7S514.

Rhodopsin mutations in a Scottish retinitis pigmentosa population, including a novel splice site mutation in intron four.

Retinitis pigmentosa (RP) is the name given to a group of disorders, both clinically and genetically heterogeneous, that primarily affect the photoreceptor function of the eye. Mutations in the genes encoding for rhodopsin, RDS-peripherin, or the beta subunit of the cGMP phosphodiesterase enzyme can be responsible for the phenotype. In this study the rhodopsin gene has been screened for mutations in a panel of RP individuals and five different sequence changes have been detected to date in three dominantly inherited and two unclassified families. One of these, a base substitution in the 3'UTR, has not yet been confirmed as disease specific, while three missense substitutions have previously been reported and are likely to be responsible for the phenotype. The fifth change, a base substitution at the intron 4 acceptor splice site, represents a novel mutation and is assumed to be the causative mutation.

Glutathione increases interleukin-2 production in human lymphocytes.

It is known that glutathione (GSH) has an immunological effect on several features of the immune system. The present study investigated the effects of GSH on interleukin-2 (IL-2) production from normal human peripheral blood lymphocytes (PBL). The results showed that both exogenous GSH and 2-mercaptoethanol (2-ME) significantly increased intracellular GSH levels after PBL were incubated with both agents. IL-2 production from PBL was markedly increased at the presence of exogenous GSH (0.5-8 mmol/l) or 2-ME (12.5-50 mumol/l) which corresponded to 1.57-2.82 nmol/10(6) cells and 1.41 - 1.80 nmol/10(6) cells of intracellular concentrations of GSH, respectively. However, IL-2 production seemed to reach a steady level when exogenous GSH concentrations in cell culture were between 2 and 8 mmol/l. The findings also showed that there was a positive correlation between the IL-2 concentrations and intracellular GSH levels. This study indicated that both exogenous GSH and 2-ME were able to elevate intracellular GSH levels and the increased intracellular GSH could increase IL-2 production in vitro. It is suggested that GSH may exert its effects on the immune system via the regulation of IL-2 synthesis.

Pathology of hereditary retinal degeneration associated with hypobetalipoproteinemia.

BACKGROUND: The clinical features and previously unreported ocular pathology in a case of heterozygous hypobetalipoproteinemia (HBL) associated with a pigment epitheliopathy are documented. Night blindness developed in a white woman with familial heterozygous HBL (cholesterol and low-density lipoprotein levels < 5% of normal) at 51 years of age. Ophthalmoscopy showed bilateral symmetric depigmentation at the posterior pole with pigment clumping and pavingstone configuration in the periphery. By the time the patient died, at 75 years of age, vision had deteriorated to hand motions. METHODS: One eye was removed 2 hours postmortem for light and electron microscopic study. RESULTS: The photoreceptors were absent, and the outer nuclear layer was replaced by glial cells throughout most of the retina, but there was some focal photoreceptor preservation in isolated regions. The outstanding feature was a massive deposition of basal linear deposit which was calcified in segments and which contained macrophages and the processes of glial cells: trilaminar bodies and melanin granules were identified in the macrophages. The remaining retinal pigment epithelial cells contained melanin but very little lipofuscin: intraretinal migration was minimal. CONCLUSIONS: The authors postulate that the pigment epitheliopathy associated with HBL is an abiotrophy in which photoreceptor discs are unable to regenerate due to locally disordered metabolism resulting from or acting in concert with the pigment epitheliopathy.

An increased incidence of apolipoprotein E2/E2 and E4/E4 in retinitis pigmentosa.

Previous studies from our laboratory have shown that retinitis pigmentosa (RP), a family of hereditary retinal degenerations, is often accompanied by abnormal levels of cholesterol or polyunsaturated fatty acids. The requirement of the retina for n-3 fatty acids is well known, and a defect in the supply of these lipids (e.g., by apolipoproteins) could affect the course of the disease. The present study confirms and extends a report on apolipoprotein E (apo E) isoforms in German RP patients [Jahn, Oette, Esser, Bergmann, and Leiss, (1987) Ophthalmic Res. 19, 285-288] which showed a tenfold increased frequency of the E2/E2 phenotype compared to the average German population. In our study, apo E phenotypes were determined in the probands of 100 Scottish RP families. The findings revealed a 4-fold increase in the incidence of E2/E2 and an 8-fold increase in E4/E4 compared to a Scottish control population. These increases were statistically significant at the P < 0.05 and P < 0.01 levels, respectively. To investigate the possibility that some of these apparent E2/E2 or E4/E4 phenotypes might actually be new apo E mutations, we examined the behavior of the apo E on sodium dodecyl sulfate-polyacrylamide gels (E2 migrates anomalously) and on isoelectric focusing gels following cysteamine modification of cysteines. These studies showed that two RP patients possibly had new apo E mutations, though amino-terminal sequence analysis revealed no changes in the sequence of the first 19 residues; further sequence analysis is obviously warranted.

Specific radioimmunoassay to investigate rod outer segment phagocytosis by retinal pigment epithelium in vitro.

A sensitive radioimmunoassay has been developed which allows rapid quantitation of rod outer segment (ROS) phagocytosis by retinal pigment epithelial (RPE) explants in vitro. It involves the use of an antiopsin antiserum, in conjunction with 125I-protein A as a second antibody, and utilizes permeabilization with ethanol to distinguish between the binding and ingestion phases of phagocytosis. This procedure will be used in the future to investigate potential regulatory factors of ROS phagocytosis by retinal pigment epithelium and to evaluate animal models of retinal degeneration.

Experimental autoimmune uveoretinitis and pinealitis induced by interphotoreceptor retinoid-binding protein and S-antigen: induction of intraretinal and subretinal neovascularization.

Experimental autoimmune uveoretinitis (EAU) and pinealitis were induced in Lewis rats following hind foot pad injection of interphotoreceptor retinoid-binding protein (IRBP) or S-antigen. A comparison is made in this study of the in vivo and histological changes in uveoretinitis and pinealitis induced by administering similar doses of highly-purified IRBP and S-antigen emulsified in complete Freund's adjuvant (CFA). The time of onset of ocular inflammation after inoculation was slightly later in S-antigen (14-18 days) as compared with IRBP-inoculated animals (10-14 days), while the severity of the inflammation was lower in the latter group. The distribution of inflammation in the anterior segment was similar in both the S-antigen and IRBP sensitized animals but there was major variation in the location of the posterior segment disease. Vasculitis was a predominant feature of IRBP induced disease while chorioretinitis and photoreceptor destruction was more prominent in the S-antigen sensitized animals. A striking feature of this study is that both antigens induced intraretinal and subretinal neovascularization, an observation which has not been reported previously. Inflammation was induced in all pineal glands and as with EAU the severity was closely related to the type of antigen inoculated.

D-[3H]galactose incorporation into glycogen in retinal cone cells.

Previous studies have shown that bovine retinas incubated with [3H]galactose incorporated it, unmodified, into large molecules. Light and electron microscope autoradiography showed a significant proportion of the label to be in cone inner segments, and pulse-chase studies showed it was subsequently transported to the synaptic pedicles. In this report, evidence is presented to show that the galactose-labelled macromolecules are resistant to hydrolysis by proteolytic enzymes, testicular hyaluronidase, chondroitinase ABC, beta-glucosidase and beta-glucuronidase, but are readily degraded by alpha-amylase and beta-galactosidase, and to a lesser extent by beta-amylase. Treatment with alpha-amylase also leads to specific removal of radioactivity from cone inner segments and pedicles, as judged by light-microscopic autoradiography. These studies appear to indicate that the cone-specific galactose label is in glycogen or glycogen-like molecules.

Effect of glycoconjugates on rod outer segment phagocytosis by retinal pigment epithelial explants in vitro assessed by a specific double radioimmunoassay procedure.

Rod outer segment (ROS) phagocytosis by explanted bovine retinal pigment epithelium (RPE) was evaluated by a procedure using an indirect double radioimmunoassay which distinguished between ROS attached to the RPE cell surface and those which had been ingested. This approach has been used to investigate the effect of a variety of glycoconjugates on the phagocytic process. Inclusion of the glycosaminoglycans (GAGs) chondroitin sulphate type-A (CS-A) and type-C (CS-C), hyaluronic acid (HA) or dermatan sulphate (DS) in the incubation medium significantly inhibited the ingestion phase of ROS phagocytosis, whereas the binding phase was inhibited to a lesser extent. The interphotoreceptor matrix (IPM), containing these GAGs as part of proteoglycans, also had an inhibitory effect on phagocytosis. The free monosaccharides mannose, fucose and galactose all stimulated the ingestion of ROS by RPE cells. These findings support the suggestion that glycoconjugates may have a physiological role in the photoreceptor renewal process.

A specific ELISA using purified opsin, for studying autoimmunity in retinal diseases.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed to measure nanogram quantities of rhodopsin or its apoprotein, opsin, in bovine retinal rod outer segment (ROS) preparations. Anti-opsin anti-sera could detect as little as 4 ng of purified opsin or of opsin in ROS preparations. The purified opsin was prepared by quantitative elution from a preparative polyacrylamide gel, and showed higher immunoreactivity with anti-opsin than did ROS when the same amount (per weight) of protein was allowed to bind in the wells of the ELISA plates. The effect of the ionic detergent SDS (sodium dodecyl sulphate) on the immunoreactivity and antigen binding to the ELISA wells was studied. Concentrations of 0.1% SDS and above reduced the apparent binding of opsin with anti-opsin when examined by ELISA. This may have been because the negatively charged SDS reduced the efficiency of the antigen coating process, or because changes in the epitopes' conformations made them less recognisable by the corresponding antibodies. A similar ELISA system using a specific anti-S-antigen anti-serum allowed the detection of even very small amounts (nanograms) of S-antigen in ROS preparations. The presence of S-antigen in ROS preparations was confirmed by immunoblotting. Thus purified opsin is preferable to ROS for ELISA tests of autoimmunity to rhodopsin in retinal diseases. These sensitive ELISA techniques could be used to examine the presence of minute amounts of rhodopsin, opsin or S-antigen in different retinal preparations.

Short-term organ culture of the retinal pigment epithelium in microtitration plates: ultrastructural studies.

Microtitration plates were used to culture simultaneously multiple, small (6 mm diameter) explants of bovine retinal pigment epithelium (RPE). Evaluation of tissue by light microscopy and by scanning and transmission electron microscopy after various incubation periods up to 6 h showed that RPE maintained in this system retains near normal morphology. Initially, the explanted RPE lacks apical microvilli, but during the first 2-3 h in culture recovery of apical microvilli occurs. The results suggest that the system is suitable for short-term maintenance of RPE for experimental purposes. Moreover, the ability to culture up to 16 explants from one bovine eye aids statistical evaluation of RPE behaviour under varying experimental conditions.